The broad outlines of the secretory pathway were established by pioneering EM and cell fractionation experiments conducted by George Palade in the 1960s. Beginning in the mid 1970s and early 80s, my laboratory isolated a series of conditionally lethal, temperature-sensitive mutations that block secretion at one of several sequential stages along the pathway established by Palade. Concurrently, James Rothman’s laboratory established a cell-free reaction that reproduced vesicular traffic within the Golgi apparatus, and several of the proteins he isolated with this functional assay matched the Sec proteins we identified. Using a cell-free vesicle budding reaction, my laboratory isolated a complex of Sec proteins that comprise a coat, COPII, responsible for cargo vesicle traffic from the endoplasmic reticulum.

Autophagosomes mature by the addition of membrane material from various intracellular sources and the attachment of peripheral proteins that remain bound through a covalent lipidation reaction. However, the origin and the mechanism of generation of the pre-autophagic membrane are poorly understood. We addressed this question with the development and analysis of a cell-free reaction that reproduces the lipidation of a major peripheral autophagosomal protein, LC3. A crude membrane fraction isolated from cells deficient in lipidation was mixed with cytosol harvested from normal cells that were untreated or subjected to a stress regimen known to induce autophagy. On addition of ATP, incubation of the mixture resulted in the formation of lipidated LC3. The reaction requires both membranes and cytosol, and was found to be stimulated 2- to 5-fold when the cytosol was taken from stress-induced cells. Autophagosome maturation requires a class III PI-3 kinase (VPS34 homolog); LC3 lipidation in our cell-free reaction is inhibited by inhibitors of this kinase, and by the addition of a peptide containing a PI3P-bnding sequence, the FYVE domain. Using cell fractionation techniques we have identified the ER-Golgi intermediate (ERGIC) compartment as the major site for lipidation of LC-3. This cell-free reaction may now be used to understand the molecular mechanism of autophagosome maturation.